Hoogsteen Research Articles

Adenine-based receptor for dicarboxylic acids

The adenine-based fluorescent receptor 1 was designed and synthesized for the selective recognition of dicarboxylic acids in CH 3CN. The recognition takes place through the Hoogsteen binding site of adenine with concomitant PET quenching of the anthracene moiety. The carboxylic acid binding to 1 was investigated by 1H NMR, X-ray, UV–vis, and fluorescence spectroscopic methods. The Hoogsteen (HG) cleft of receptor 1 is found to be selective for glutaric acid.

Hydrogen Bonding of 7,8-Dihydro-8-oxodeoxyguanosine with a Charged Residue in the Little Finger Domain Determines Miscoding Events in Sulfolobus solfataricus DNA Polymerase Dpo4

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) has been shown to catalyze bypass of 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) in a highly efficient and relatively accurate manner. Crystal structures have revealed a potential role for Arg(332) in stabilizing the anti conformation of the 8-oxoG template base by means of a hydrogen bond or ion-dipole pair, which results in an increased enzymatic efficiency for dCTP insertion and makes formation of a Hoogsteen pair between 8-oxoG and dATP less favorable. Site-directed mutagenesis was used to replace Arg(332) with Ala, Glu, Leu, or His in order to probe the importance of Arg(332) in accurate and efficient bypass of 8-oxoG. The double mutant Ala(331)Ala(332) was also prepared to address the contribution of Arg(331). Transientstate kinetic results suggest that Glu(332) retains fidelity against bypass of 8-oxoG that is similar to wild type Dpo4, a result that was confirmed by tandem mass spectrometric analysis of full-length extension products. A crystal structure of the Dpo4 Glu(332) mutant and 8-oxoG:C pair revealed water-mediated hydrogen bonds between Glu(332) and the O-8 atom of 8-oxoG. The space normally occupied by Arg(332) side chain is empty in the crystal structures of the Ala(332) mutant. Two other crystal structures show that a Hoogsteen base pair is formed between 8-oxoG and A in the active site of both Glu(332) and Ala(332) mutants. These results support the view that a bond between Arg(332) and 8-oxoG plays a role in determining the fidelity and efficiency of Dpo4-catalyzed bypass of the lesion.

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 14

AbstractThe self‐complementary tetrameric propargyl triols 8, 14, 18, and 21 were synthesized to investigate the duplex formation of self‐complementary, ethynylene‐linked UUAA, AAUU, UAUA, and AUAU analogues with integrated bases and backbone (ONIBs). The linear synthesis is based on repetitive Sonogashira couplings and C‐desilylations (34–72% yield), starting from the monomeric propargyl alcohols 9 and 15 and the iodinated nucleosides 3, 7, 11, and 13. Strongly persistent intramolecular H‐bonds from the propargylic OH groups to N(3) of the adenosine units prevent the gg‐type orientation of the ethynyl groups at C(5′). As such, an orientation is required for the formation of cyclic duplexes, this H‐bond prevents the formation of duplexes connected by all four base pairs. However, the central units of the UAUA and AAUU analogues 18 and 14 associate in CDCl3/(D6)DMSO 10 : 1 to form a cyclic duplex characterized by reverse Hoogsteen base pairing. The UUAA tetramer 8 forms a cyclic UU homoduplex, while the AUAU tetramer 21 forms only linear associates. Duplex formation of the O‐silylated UUAA and AAUU tetramers is no longer prevented. The self‐complementary UUAA tetramer 22 forms Watson–Crick‐ and Hoogsteen‐type base‐paired cyclic duplexes more readily than the sequence‐isomeric AAUU tetramer 23, further illustrating the sequence selectivity of duplex formation.

Biophysical Characterization of the Human Telomeric (TTAGGG)4 Repeat in a Potassium Solution

Quadruplex structures arise from four coplanar G bases arranged in a Hoogsteen base pairing motif to create a central pore that can coordinate cations. The termini of eukaryotic chromosomes contain structures, known as telomeres, which are capable of forming quadruplex structures. Quadruplexes have been implicated in a variety of disease states, including cancer. The literature seems to agree that the human telomeric repeat containing four stretches of three guanines displays conformational states that are different in the presence of Na+ and K+ and an unknown number of species involved in the quadruplex to single strand transition. Using circular dichroism spectroscopy, differential scanning calorimetry, and singular-value decomposition, the number of species present in the dissociation process is assessed. The results indicate that three species exist in equilibria during the melting process. We present a model for the heat-induced denaturation from the folded to the unfolded state, whereby the hybrid parallel-antiparallel quadruplex undergoes a transition to an unknown intramolecular intermediate followed by a transition to a single strand.

Self-Assembling Supramolecular Complexes by Single-Stranded Extension from Plasmid DNA

Self-assembling supramolecular complexes are of great interest for bottom-up research like nanotechnology. DNA is an inexpensive building block with sequence-specific self-assembling capabilities through Watson-Crick and/or Hoogsteen base pairing and could be used for applications in surface chemistry, material science, nanomechanics, nanoelectronics, nanorobotics, and of course in biology. The starting point is usually single-stranded DNA, which is rather easily accessible for base pairing and duplex formation. When long stretches of double-stranded DNA are desirable, serving either as genetic codes or electrical wires, bacterial expansion of plasmids is an inexpensive approach with scale-up properties. Here, we present a method for using double-stranded DNA of any sequence for generating simple structures, such as junctions and DNA lattices. It is known that supercoiled plasmids are strand-invaded by certain DNA analogs. Here we add to the complexity by using "Self-assembling UNiversal (SUN) anchors" formed by DNA analog oligonucleotides, synthesized with an extension, a "sticky-end" that can be used for further base pairing with single-stranded DNA. We show here how the same set of SUN anchors can be utilized for gene therapy, plasmid purification, junction for lattices, and plasmid dimerization through Watson-Crick base pairing. Using atomic force microscopy, it has been possible to characterize and quantify individual components of such supra-molecular complexes.

Alternate PNA‐DNA chimeras (PNA‐DNA)n: Synthesis, binding properties and biological activity

Peptide nucleic acids (PNAs) are oligonucleotide mimics in which the sugar-phosphate backbone has been replaced by a pseudo-peptide backbone. Among PNA-based molecules, PNA-DNA conjugates characterized by tracts of DNA bound to N and/or C terminus of PNA are very soluble in aqueous media, are able to recognize exclusively single strands of DNA and RNA in antiparallel fashion, activate RNAse H, bind to transcription factors and are more stable than DNA to nucleases degradation. Very little information is available on chimeras constituted of alternating monomers of PNA and DNA. In this article, we describe a simple fully automated strategy for the synthesis of 6-mer and 10-mer alternate PNA-DNA chimeras consisting of polythymine oligomers, stability assays in fetal calf serum, UV and CD studies of the single strand alternate chimeras and of alternate chimera/DNA and alternate chimera/RNA duplexes. Evidences supporting the formation of duplex hybrids were found. Furthermore, the ability of forming Hoogsteen base pairing with duplex DNA was investigated. Finally, we tested the ability of the PNA-DNA alternates in (a) interfering with reverse transcription of eukaryotic mRNA and (b) inhibiting DNA-protein interactions.

Betaine, Dimethyl Sulfoxide, and 7-Deaza-dGTP, a Powerful Mixture for Amplification of GC-Rich DNA Sequences

Currently, polymerase chain reaction is the most used technique in many laboratories for either diagnostic or molecular biology purposes. Despite the large number of DNA sequences that can be easily analyzed, some GC-rich sequences are refractory to amplification due to the formation of secondary intramolecular structures. To overcome this problem, several molecules have been described to improve polymerization. Here we show that a combination of three additives--betaine, dimethyl sulfoxide, and 7-deaza-dGTP--was essential to achieve amplification of DNA sequences of three disease genes showing a GC content ranging from 67 to 79%.

Factors regulating thermodynamic stability of DNA structures under molecular crowding conditions

The condition in a living cell is molecularly crowded with various biomolecules. The total concentration of the biomolecules inside Escherichia coli is in the range of 300-400 g/L. This is distinct from typical biomolecular concentrations of less than 1g/L, which is generally used for experiments in vitro. Here, we analyzed quantitatively the effects of molecular crowding on the thermodynamics of antiparallel G-quadruplex formation via Hoogsteen base pairs and of antiparallel hairpin-looped duplex (HP duplex) formation via Watson-Crick base pairs. The free energy changes for G-quadruplex and duplex formations decreased and increased when the concentration of poly(ethylene glycol) 200 was increased from 0 to 40 wt%, respectively. These results showed that the antiparallel G-quadruplex is stabilized under molecular crowding conditions but the HP duplex is destabilized.

Synthesis and Pairing Properties of α‐Tricyclo‐DNA

We describe the synthesis of the phosphoramidite building blocks of alpha-tricyclo-DNA (alpha-tc-DNA) covering all four natural bases, starting from the already known corresponding alpha-tc-nucleosides. These building blocks were used for the preparation of three alpha-tc-oligonucleotide 10-mers representing a homopurine, a homopyrimidine, and a mixed purine/pyrimidine base sequence. The base-pairing properties with complementary parallel and antiparallel oriented DNA and RNA were studied by UV-melting analysis and CD spectroscopy. We found that alpha-tc-DNA binds preferentially to parallel nucleic acid complements through Watson-Crick duplex formation, with a preference for RNA over DNA. In comparison with natural DNA, alpha-tc-DNA shows equal to enhanced affinity to RNA and also pairs to antiparallel DNA or RNA complements, although with much lower affinity. In the mixed-base sequence these antiparallel duplexes are of the reversed Watson-Crick type, while in the homopurine/homopyrimidine sequences Hoogsteen and/or reversed Hoogsteen pairing is observed. Antiparallel duplex formation of two alpha-tc-oligonucleotides was also observed, although the thermal stability of this duplex was surprisingly low. The base-pairing properties of alpha-tc-DNA are discussed in the context of alpha-DNA, alpha-RNA, and alpha-LNA.

The Paperclip Triplex: Understanding the Role of Apex Residues in Tight Turns

In this study, we investigate the role of the apex nucleotides of the two turns found in the intramolecular “paperclip” type triplex DNA formed by 5′-TCTCTCCTCTCTAGAGAG-3′. Our previously published structure calculations show that residues C 7-A 18 form a hairpin turn via Watson-Crick basepairing and residues T 1-C 6 bind into the major groove of the hairpin via Hoogsteen basepairing resulting in a broad turn of the T 1-T 12 5′-pyrimidine section of the DNA. We find that only the C 6C 7/G 18 apex triad (and not the T 12A 13/T 1 apex triad) is required for intramolecular triplex formation, is base independent, and occurs whether the purine section is located at the 5′ or 3′ end of the sequence. NMR spectroscopy and molecular dynamics simulations are used to investigate a bimolecular complex (which retains only the C 6C 7/G 18 apex) in which a pyrimidine strand 5′- TCTCTCCTCTCT-3′ makes a broad fold stabilized by the purine strand 5′-AGAGAG-3′ via Watson Crick pairing to the T 8-T 12 and Hoogsteen basepairing to T 1-T 5 of the pyrimidine strand. Interestingly, this investigation shows that this 5′-AGAGAG-3′ oligo acts as a new kind of triplex forming oligonucleotide, and adds to the growing number of triplex forming oligonucleotides that may prove useful as therapeutic agents.

Towards a better understanding of the unusual conformations of the alternating guanine–adenine repeat strands of DNA

Alternating guanine-adenine strands of DNA are known to self-associate into a parallel-stranded homoduplex at neutral pH, fold into an ordered single-stranded structure at acid pH, and adopt yet another ordered single-stranded conformer in aqueous ethanol. The unusual conformers melt cooperatively and exhibit distinct circular dichroism spectra suggestive of a substantial conformational order, but their molecular structures are not known yet. Here, we have probed the molecular structures using guanine and adenine analogs lacking the N7 atom, and thus unable of Hoogsteen pairing, or those restrained in the less-frequent syn glycosidic orientation. The studies showed that the syn glycosidic orientation of dA residues promoted the neutral homoduplex, whereas the syn orientation of dG was incompatible with the homoduplex. In addition, Hoogsteen pairing of dA seemed to be a crucial property of the homoduplex whereas dG did not pair in this way. The situation was the same in both single-stranded conformers with the dG residues. On the other hand, the presence of N7 was important with dA but its syn geometry was not favorable. The present data can be used as restraints to model the unusual molecular structures of the alternating guanine-adenine strands of DNA.

Role of Hoogsteen Edge Hydrogen Bonding at Template Purines in Nucleotide Incorporation by Human DNA Polymerase ι

Human DNA polymerase iota (Pol iota) differs from other DNA polymerases in that it exhibits a marked template specificity, being more efficient and accurate opposite template purines than opposite pyrimidines. The crystal structures of Pol iota with template A and incoming dTTP and with template G and incoming dCTP have revealed that in the Pol iota active site, the templating purine adopts a syn conformation and forms a Hoogsteen base pair with the incoming pyrimidine which remains in the anti conformation. By using 2-aminopurine and purine as the templating residues, which retain the normal N7 position but lack the N(6) of an A or the O(6) of a G, here we provide evidence that whereas hydrogen bonding at N(6) is dispensable for the proficient incorporation of a T opposite template A, hydrogen bonding at O(6) is a prerequisite for C incorporation opposite template G. To further analyze the contributions of O(6) and N7 hydrogen bonding to DNA synthesis by Pol iota, we have examined its proficiency for replicating through the (6)O-methyl guanine and 8-oxoguanine lesions, which affect the O(6) and N7 positions of template G, respectively. We conclude from these studies that for proficient T incorporation opposite template A, only the N7 hydrogen bonding is required, but for proficient C incorporation opposite template G, hydrogen bonding at both the N7 and O(6) is an imperative. The dispensability of N(6) hydrogen bonding for proficient T incorporation opposite template A has important biological implications, as that would endow Pol iota with the ability to replicate through lesions which impair the Watson-Crick hydrogen bonding potential at both the N1 and N(6) positions of templating A.

Hoogsteen base pair formation promotes synthesis opposite the 1,N6-ethenodeoxyadenosine lesion by human DNA polymerase ι

The 1,N6-ethenodeoxyadenosine (epsilon dA) lesion is promutagenic and has been implicated in carcinogenesis. We show here that human Pol iota, a Y-family DNA polymerase, can promote replication through this lesion by proficiently incorporating a nucleotide opposite it. The structural basis of this action is rotation of the epsilon dA adduct to the syn conformation in the Pol iota active site and presentation of its 'Hoogsteen edge' for hydrogen-bonding with incoming dTTP or dCTP. We also show that Pol zeta carries out the subsequent extension reaction and that efficiency of extension from epsilon dA x T is notably higher than from epsilon dA x C. Together, our studies reveal for the first time how the exocyclic epsilon dA adduct is accommodated in a DNA polymerase active site, and they show that the combined action of Pol iota and Pol zeta provides for efficient and error-free synthesis through this potentially carcinogenic DNA lesion.

Hydration Regulates Thermodynamics of G-Quadruplex Formation under Molecular Crowding Conditions

The effect of molecular crowding on the structure and stability of biomolecules has become a subject of increasing interest because it can clarify how biomolecules behave under cell-mimicking conditions. Here, we quantitatively analyzed the effects of molecular crowding on the thermodynamics of antiparallel G-quadruplex formation via Hoogsteen base pairs and of antiparallel hairpin-looped duplex (HP duplex) formation via Watson-Crick base pairs. The free energy change at 25 degrees C for G-quadruplex formation decreased from -3.5 to -5.5 kcal mol(-1) when the concentration of poly(ethylene glycol) 200 was increased from 0 to 40 wt %, whereas that of duplex formation increased from -9.8 to -6.9 kcal mol(-1). These results showed that the antiparallel G-quadruplex is stabilized under molecular crowding conditions, but that the HP duplex is destabilized. Moreover, plots of stability (ln K(obs)) of the DNA structures versus water activity (ln a(w)) demonstrated that the ln K(obs) for G-quadruplex formation decreased linearly as the ln a(w) increased, whereas that for duplex formation increased linearly with the increase in ln a(w), suggesting that the slope approximately equals the number of water molecules released or taken up during the formation of these structures. Thus, molecular crowding affects the thermodynamics of DNA structure formation by altering the hydration of the DNA. The stabilization of the DNA structures with Hoogsteen base pairs and destabilization of DNA structures with Watson-Crick base pairs under molecular crowding conditions lead to structural polymorphism of DNA sequences regulated by the state of hydration.

Kinetic Evidence for Inefficient and Error-prone Bypass across Bulky N2-Guanine DNA Adducts by Human DNA Polymerase ι

DNA polymerase (pol) iota has been proposed to be involved in translesion synthesis past minor groove DNA adducts via Hoogsteen base pairing. The N2 position of G, located in minor groove side of duplex DNA, is a major site for DNA modification by various carcinogens. Oligonucleotides with varying adduct size at G N2 were analyzed for bypass ability and fidelity with human pol iota. Pol iota effectively bypassed N2-methyl (Me)G and N2-ethyl(Et)G, partially bypassed N2-isobutyl(Ib)G and N2-benzylG, and was blocked at N2-CH2(2-naphthyl)G (N2-NaphG), N2-CH2(9-anthracenyl)G (N2-AnthG), and N2-CH2(6-benzo[a]pyrenyl)G. Steady-state kinetic analysis showed decreases of kcat/Km for dCTP insertion opposite N2-G adducts according to size, with a maximal decrease opposite N2-AnthG (61-fold). dTTP misinsertion frequency opposite template G was increased 3-11-fold opposite adducts (highest with N2-NaphG), indicating the additive effect of bulk (or possibly hydrophobicity) on T misincorporation. N2-IbG, N2-NaphG, and N2-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (S(p)-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) opposite N2-EtG and N2-AnthG (but not G) suggest that the chemistry step is largely interfered with by adducts. Severe inhibition of polymerization opposite N2,N2-diMeG compared with N2-EtG by pol eta but not by pol iota is consistent with Hoogsteen base pairing by pol iota. Thus, polymerization by pol iota is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol iota may play a limited role in translesion synthesis on bulky N2-G adducts in cells.

An Incoming Nucleotide Imposes an anti to syn Conformational Change on the Templating Purine in the Human DNA Polymerase-ι Active Site

Substrate-induced conformational change of the protein is the linchpin of enzymatic reactions. Replicative DNA polymerases, for example, convert from an open to a closed conformation in response to dNTP binding. Human DNA polymerase-iota (hPoliota), a member of the Y family of DNA polymerases, differs strikingly from other polymerases in its much higher proficiency and fidelity for nucleotide incorporation opposite template purines than opposite template pyrimidines. We present here a crystallographic analysis of hPoliota binary complexes, which together with the ternary complexes show that, contrary to replicative DNA polymerases, the DNA, and not the polymerase, undergoes the primary substrate-induced conformational change. The incoming dNTP "pushes" templates A and G from the anti to the syn conformation dictated by a rigid hPoliota active site. Together, the structures posit a mechanism for template selection wherein dNTP binding induces a conformational switch in template purines for productive Hoogsteen base pairing.

Impact of the Guanidinium Group on Hybridization and Cellular Uptake of Cationic Oligonucleotides

The grafting of cationic groups to synthetic oligonucleotides (ONs) in order to reduce the charge repulsion between the negatively charged strands of a duplex or triplex, and consequently to increase a complex's stability, has been extensively studied. Guanidinium groups, which are highly basic and positively charged over a wide pH range, could be an efficient ON modification to enhance their affinity for nucleic acid targets and to improve cellular uptake. A straightforward post-synthesis method to convert amino functions attached to ONs (on sugar, nucleobase or backbone) into guanidinium tethers has been perfected. In comparison to amino groups, such cationic groups anchored to alpha-oligonucleotide phosphoramidate backbones play important roles in duplex stability, particularly with RNA targets. This high affinity could be explained by dual recognition resulting from Watson-Crick or Hoogsteen base pairing combined with cationic/anionic backbone recognition between strands involving H-bond formation and salt bridging. Molecular-dynamics simulations corroborate interactions between the cationic backbones of the alpha-ONs and the anionic backbones of the nucleic acid targets. Moreover, ONs with guanidinium modification increased cellular uptake relative to negatively charged ONs. The cellular localization of these new cationic phosphoramidate ONs is mainly cytoplasmic. The uptake of these ON analogues might occur through endocytosis.

Hoogsteen base-pairing revisited: Resolving a role in normal biological processes and human diseases

For a long time since the discovery of an alternative type of hydrogen bonding between adenine and thymidine, termed Hoogsteen base-pairing, its biological role remained elusive. Recent experiments provide compelling evidence that Hoogsteen base pairs manifest in a gamut of nuclear processes encompassing gene expression, replication, recombination, and telomere length maintenance. An increasing number of proteins that have been shown to bind, unwind or cleave G-quadruplexes or triplexes with high specificity underscore their biological significance. In humans, the absence of these cellular factors or their dysfunction leads to a wide spectrum of genetic diseases including cancer, neurodegenerative syndromes, and a myriad of other disorders. Thus, development of clinically useful compounds that target G-quadruplexes or triplexes, and interfere with specific cellular processes, provides considerable promise for successful and improved treatment of human diseases.

Efficient and High Fidelity Incorporation of dCTP Opposite 7,8‐Dihydro‐8‐oxodeoxyguanosine by Sulfolobus solfataricus DNA Polymerase Dpo4

DNA polymerases insert dATP opposite the oxidative damage product 7,8 dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of > 90% with some polymerases. Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 25-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events with dCTP was similar for G and 8-oxoG templates. Mass spectral analysis of extended DNA primers showed ≥ 95% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured Kd,dCTP was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The Ea for dCTP insertion opposite 8-oxoG was lower than opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca2+. The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with the 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5′ C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays.

Structure of Purine−Purine Mispairs during Misincorporation and Extension by Escherichia coli DNA Polymerase I

The mechanism by which purine-purine mispairs are formed and extended was examined with the high-fidelity Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease activity inactivated. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. A decrease in rate associated with a 7-deazapurine substitution would suggest that the nucleotide is in a syn conformation in a Hoogsteen base pair with the opposite base. During mispair formation, the k(pol)/K(d) values for the insertion of dATP opposite A (dATP/A) as well as dATP/G and dGTP/G were decreased greater than 10-fold with the deazapurine in the dNTP. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the syn conformation and the template base in the anti conformation. During mispair extension, the only decrease in k(pol)/K(d) was associated with the G/G base pair in which 7-deazaguanine was in the template strand. These results as well as previous results [McCain et al. (2005) Biochemistry 44, 5647-5659] in which a hydrogen bond was found between the 3-position of guanine at the primer terminus and Arg668 during G/A and G/G mispair extension indicate that the conformation of the purine at the primer terminus is in the anti conformation during mispair extension. These results suggest that purine-purine mispairs are formed via a Hoogsteen geometry in which the dNTP is in the syn conformation and the template is in the anti conformation. During extension, however, the conformation of the primer terminus changes to an anti configuration while the template base may be in either the syn or anti conformations.